这项工作是由跨学科临床研究中心（IZKF）明斯特（种子03/12，SB）的支持。

1，总评的小鼠实验<ol><li>利用老鼠实验都应该按照各自的机构动物护理和使用委员会的指导下进行。 </li><li>保持鼠标无病原体条件下，使他们能够获得食物和水自由采食 。注意：使用年龄和性别匹配的小鼠在实验组是很重要的，因为对疾病的易感性可能与年龄和性别而有所不同。 </li><li>取决于所选择的实验条件下，一个假免疫的对照组可以被认为是其中MOG 35-55肽被替换或者PBS无抗原或nonencephalitogenic肽。 </li><li>请考虑方法问题之前，要启动试验（见下文）。我们建议涉及一个或两个盲观察员EAE得分。 </li></ol> 2，准备MOG 35-55乳液<ol><li>加入200μl1:1的比例MO的摹35-55肽溶液和终审法院应注射到每只小鼠。有粘性乳液的一些损失，同时准备和注射。因此，准备所需量的1.5倍。计算该乳液和除以2为MOG 35-55肽溶液和CFA的所需量的总体积。 </li><li>在DDH 2 O稀释冻干MOG 35-55至2毫克/毫升的最终浓度。我们通常使用200微克MOG每只小鼠35-55肽。这个量被包含在100μl原液。肽溶液应储存于-20℃。 </li><li>放置1瓶干燥处理结核杆菌H37RA（100毫克）的内容转换成一个迫击炮。 <ol><li>地面用研钵和杵，得到细粉末。 </li><li>加入10毫升弗氏不完全佐剂，以获得可被保存在4℃的10毫克/毫升CFA原液</li><li>前免疫，终审法院稀释原液机智ħIFA至2毫克/毫升的最终浓度。每次使用悬浮颗粒物质，并考虑粘稠溶液在实验准备一些体积损失之前，调匀。 </li><li>用MOG 35-55肽溶液混合1点01分为止的1毫克的最终浓度/毫升为止。 注意：热灭活的结核分枝杆菌刺激先天免疫应答。避免吸入，食入和皮肤和眼睛接触。 </li></ol></li><li>预冷冰的解决方案。 <ol><li>绘制升1毫升2毫克/毫升终审法院和1ml 2毫克/毫升MOG 35-55解成两个2毫升注射器。根据免疫动物的数目计算出必要的注射器的数量。 注意：严格避免配制佐剂拼接过程中，因为它可能会导致肉芽肿或诱发自身免疫反应。 </li><li>使用27G的套管为MOG 35-55溶液和20 G套管的终审法院。避免产生气泡，并与在连接两个注射器重稀土单向阀。 </li><li>发送乳液从一个针筒到另一调匀至少10分钟作为一个很好的乳化是关键的一步。近乎关闭三通阀来支持乳化。该解决方案应该是白色的，僵硬和粘性与相没有分离。 </li><li>乳剂可以被存储为免疫前几天。等制备的乳剂，以观察它们是否稳定后至少30分钟。前免疫，绘制解成两个注射器之一，并连接一个27G的套管。 </li></ol></li></ol>百日咳毒素的3制备<ol><li>不同量百日咳毒素可以在文献也取决于给药途径（ 如静脉注射或腹腔内）上找到。我们的实验室使用400ng的百日咳毒素在200μl的PBS腹膜内免疫的当天和两天后。注意：百日咳毒素具有多种生物学效应。避免吸入，食入，以及皮肤和眼睛接触。 </li><li>每瓶50微克百日咳毒素在500μL双蒸2 O为100微克/毫升原液。贮存于4℃。 </li><li>稀释1:50的PBS。 200微升现在含有400毫微克的PBS。准备注射器的必要量，并考虑〜100微升的附加多余的体积为每个针毂。 </li></ol> 4，动物免疫<ol><li>请参考机构动物护理和使用委员会申请所需的短期安乐死的具体标准。我们的实验室使用简短narcotization用异氟烷，而其它的协议，如氯胺酮/赛拉嗪注射或halothan也可以是可能的。等待麻醉并用前脚脚趾捏评估麻醉的水平。 </li><li>确保免疫接种是由有经验的人进行，尽量减少应激对动物，并确保最佳的免疫接种。 </li><li>注入100μL反根/ CFA乳化皮下成两个不同的站点上的每个后侧翼。保证皮肤下球茎质量的形式，应该坚持整个实验。 </li><li>注入200微升百日咳毒素intraperitonelly。 </li><li>确保个人的小鼠可以很容易地被识别为日常评估， 例如，通过对尾基部颜色标记。 </li><li>管理百日咳毒素第二剂免疫后两天。 </li></ol> 5，EAE监控<ol><li>重量和临床评分应每天评估。 EAE的发病水平通常与重量损失，这可以作为疾病活动性的一个指标。请参考机构的动物护理和使用委员会预定义标准时，个别小鼠必须取出实验。这时候减肥超过20％的初始体重，或当严重的临床症状（EAE比分7或更糟）发生应予以考虑。请参阅重各机构的动物护理和使用委员会的允许的最大比分的关产品的准则。 </li><li>当小鼠有EAE的临床症状，这确保了水瓶仍然可以达成与该食品被放置在箱地板是重要的。 </li><li>不同的评分症状都可以使用。在我们的实验室0-10级成立。 </li></ol><table border="1"><tbody><tr><td> 等级 </td><td> 临床体征 </td><td> 评论 </td></tr><tr><td> 0 </td><td>无临床症状</td><td>正常步态，尾巴的动作，并且可以提高，尾绕到一个圆形物体，如果鼠标被保持在尾的基部</td></tr><tr><td> 1 </td><td>部分柔弱的尾部</td><td>正常步态，尾巴尖下垂</td></tr><tr><td> 2 </td><td>尾部瘫痪</td><td>正常步态，尾巴下垂</td></tr><tr><td> 3 </td><td>后肢麻痹​​，运动不协调</TD&gt; <td>步态不协调，颠尾，后肢来捏回应</td></tr><tr><td> 4 </td><td>一后肢瘫痪</td><td>步态不协调的一个后肢拖，尾部颠一后肢不响应掐</td></tr><tr><td> 5 </td><td>这两个后肢瘫痪</td><td>步态不协调与两个后肢拖，尾颠，两个后肢不回应捏</td></tr><tr><td> 6 </td><td>后肢瘫痪，无力前肢</td><td>步态不协调与前肢奋斗捏后拉身体，前肢反射消失，尾部颠</td></tr><tr><td> 7 </td><td>后肢瘫痪，人们前肢瘫痪</td><td>鼠标不能动，一是前肢响应捏脚趾，尾巴颠</td></tr><tr><td> 8 </td><td>后肢瘫痪，前肢瘫痪</td><td>鼠标不能动，前肢不给脚趾捏回应，尾巴颠</td></tr><tr><td> 9 </td&gt; <td>垂死</td><td>没有运动，呼吸改变</td></tr><tr><td> 10 </td><td>死亡</td><td></td></tr></tbody></table>表1。临床评分系统。

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多种不同的EAE模型与主动免疫的协议已被描述在过去的几十年。虽然大鼠模型已被广泛使用，直到最近，老鼠是现在最流行的模式生物EAE研究工作。这种发展是其中包括由于现有的转基因小鼠的广阔和不断增长的剧目。 C57BL / 6小鼠用MOG 35-55肽免疫是分布最广的EAE模型之一，可以被视为一个可靠的，可复制和良好使用的动物模型。在许多神经免疫实验室，MOG 35-55诱导的EAE被确立为首选的模式，而其他的EAE模型被用于更具体的实验问题。 需要考虑的一个关键点是实验设置的规划，以确保EAE的实验方法上进行正确的。对于内部效度，疾病症状蒙蔽得分，强烈推荐。 EXPERimental群体应该是年龄，体重和性别匹配和鼠标应该随机分配到治疗组。实验中应始终遵循动物福利法规的规定执行。 EAE的研究往往动力不足，不考虑统计II型错误。因此，之前的实验中，样本量的计算应该被执行。必需基团的大小取决于预期的效果大小。专家进行统计分析的咨询会开始EAE的实验前，应考虑。 该协议的一些局限性需要牢记。最重要的是，AEAE数据解释是由免疫使用的佐剂和百日咳毒素具有对免疫反应两个额外的影响力的模式受到损害。还应当认为，MOG 35-55 EAE模型，主要显示了CD4 + T细胞驱动的免疫应答。 CD8 + T细胞和B细胞中发挥一个不太起眼的角色和替代方案应解决这些细胞类型时，应考虑。预计病程为急性，单相和自限性。可替换地，复发 - 缓解疾病过程也可以在替代的EAE模型来实现。该协议的另外一个重要的限制是一定的偏差朝向MS病理生理的免疫成分。在过去几年里，它已成为越来越明显，微软有很强的神经退行性组件。少突胶质细胞和神经元的结果在神经功能缺损的渐进积累的死亡。必须考虑到，在EAE模型可能不完全适合于处理有关的自身免疫性炎症的神经退行性机制的实验问题。重点是中枢神经系统的病理替代的动物模型可以被认为- 如 cuprizone模型，危及有毒脱髓鞘无外周免疫系统的参与。 所描述的协议被认为是一个基本的神经免疫实验模型，并且可以被修改为其它的应用程序。上文所述的实验步骤，可通过改变小鼠株很容易地应用到其他的EAE协议或类型和蛋白质的量（ 例如，使用PLP 139-151肽和SJL小鼠的复发-缓解EAE疾病过程是特别适合用于评估治疗对复发的影响）。所描述的协议也可用于过继转移实验（被动EAE）。在这个模型中，C57BL / 6小鼠用MOG 35-55肽和如上所述CFA。与此相反，不要求百日咳毒素。后7-15天，脾或淋巴结中分离和免疫细胞再刺激在体外用MOG 35-55肽和各种细胞因子转移到C57BL/6se小鼠的新组之前。这些受体小鼠EAE的发展早于在CLAS几天SICAL免疫。 在体外条件下可以改变的特异性免疫问题（ 例如偏振成T H 1和T H 17细胞）。 有时候，低发病率或弱的症状可能是一个实验性的挑战。故障排除有些建议是： - 疾病严重程度可以改变不同量的肽/鼠标。 - 最优CFA浓度可以从1-5毫克/毫升的不同而不同。考虑终审法院确立滴定实验时。请参阅相应的机构动物护理和使用委员会的终审法院容许浓度的各准则尽可能多的法规禁止的终审法院浓度超过2毫克/毫升。 - 不同的方法被描述用于制备乳液。如果乳化不佳被认为是possib替代方法，如涡旋1小时或超声可以考虑乐误差源。 - 年龄，性别，年份和动物设施内环境条件的季节是影响EAE的易感性的重要因素。它应当确保条件与独立实验之间。 如上所述，所提到的协议可以作为起点继EAE实验。该模型尤其适合用于分离基因的表型（ 如通过转印脑炎敲除细胞分化成野生型受体小鼠）的外周和中枢神经系统的影响，并为特定的免疫学问题，作为转移细胞的表型能充分进行表征。在EAE的研究在过去几年的最新发展，是T细胞受体的转基因小鼠。这些小鼠EAE的发展自发症状无外界影响规避佐剂接种的问题。然而，这种模式需要大量动物进行breedi纳克以确保有足够规模的团体。评价基因敲除小鼠的杂交育种，需要事先对EAE实验对比AEAE。为每只小鼠发展在不同的一天疾病症状，新颖的物质的评价可以是相当复杂的。因此，古典AEAE的神经免疫的价值仍然受到质疑。

多发性硬化（MS）是一种中枢神经系统的慢性脱髓鞘性疾病，其特征在于少突胶质细胞和神经元的结果在异种和积累临床症状的破坏。 MS被认为是中枢神经系统（CNS）的原型自身免疫性疾病和动物模型已经发展阐明其发病机制复杂的光。此外，目前的治疗仅部分有效和靶向主要疾病的炎症阶段，而神经变性成分可能是为将来的治疗的重大挑战方法1,2。 而该疾病的确切病因还不清楚，针对表位在CNS中的神经轴突的髓鞘自身免疫反应被假定惹病的发作。免疫系统，遗传脆弱性和环境因素（ 如感染，维生素D）的调节异常被认为MS的病理生理机制的影响主要方面。 三种不同类型的动物模型中对于MS的病理类型的探索正在建立的病毒：病毒模型像泰勒鼠脑脊髓炎病毒（TMEV），诱导的中毒性剂，如cuprizone模型，最后不同的实验性自身免疫性脑脊髓炎（EAE）3的变体， 4。虽然他们都模仿MS的功能，它们在潜在的病理特性，如适应性免疫系统的参与大大不同。 EAE是最常见的动物模型，因为它是非常有用的调查神经炎途径和经常作为“验证性原则”的模式为新的治疗策略，5,6的疗效。 EAE可诱发许多不同的动物（ 如小鼠，大鼠，miniswine，豚鼠，鸡，或灵长类动物）。然而，小鼠已成为最广泛使用的品种，其至少部分原因是由于扩大复杂的转基因或基因敲除小鼠7剧目。 EAE的病理生理学是基于免疫系统的抗脑特异性抗原反应。这种反应引起的炎症抗原携带结构和破坏，导致神经系统相媲美，病理特点，那些在MS患者中观察到。三种不同的方法可分为：积极诱导的EAE（AEAE;主动免疫），被动转移的EAE（pEAE;从免疫动物转移脑炎细胞），以及最近的自发的EAE小鼠模型（SEAE），允许自体免疫的研究机制没有外源性操纵。最简单的诱导模型是AEAE小鼠产生快速而稳定的结果。这种模式被认为是神经免疫动物模型的许多研究人员在外地8的“金标准”。 对于AEAE感应，动物被免疫的皮下注射由所选择的抗原乳剂和完全弗氏adjuvans（CFA）伴有腹膜内注射百日咳毒素免疫的当天和两天后。因此，髓鞘特异的T淋巴细胞在激活的外周及进入中枢神经系统迁移穿过血 - 脑屏障。在进入中枢神经系统，T细胞通过产生随后的炎性级联反应，其他细胞如单核细胞或巨噬细胞，并最终在脱髓鞘和轴突的细胞死亡9参与本地和浸润性抗原呈递细胞的活化。根据不同的免疫方案和小鼠品系的组合（ 例如 C57BL / 6，SJL / J Biozzi）和抗原（ 如髓鞘少突胶质细胞糖蛋白（MOG），髓磷脂碱性蛋白（MBP），髓鞘蛋白脂质蛋白（PLP）），所述疾病当然可以采取一种急性，慢性进行性或复发缓解病程。 35-55肽10，它产生一个单相的EAE与后9-14天，最大疾病发病和慢和局部症状恢复后约3-5天第一个症状免疫在未来的10-20天。作为单独的MOG 35-55肽的免疫原性潜力不足以诱发疾病，佐剂如CFA是必要的。据推测，这CFA的部件激活单核吞噬细胞诱导这些分子的吞噬作用和细胞因子的分泌。这将导致在抗原的存在以及它们的更有效的传输到淋巴系统的延长。 EAE诱导是通过应用程序百日咳毒素（PT）的已（其中包括）被建议来调节方便血-脑屏障和免疫应答本身11。后诱发疾病，特别必须注意的老鼠疾病症状的日常考核。

<table border="0" cellpadding="0" cellspacing="0" width="707">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Female C57BL/6 mice, age 10-12 weeks, ~20 g weight</td>
			<td style="width:205px;">e.g. Jackson Laboratory, Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MOG35-55 (MEVGWYRSPFSRVVHLYRNGK)</td>
			<td style="width:205px;">e.g. Pepceuticals Ltd</td>
			<td style="width:119px;"></td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Incomplete Freund&#8217;s adjuvant (IFA)</td>
			<td style="width:205px;">e.g. Sigma-Aldrich Co.</td>
			<td style="width:119px;">F5506</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringes, 1 ml with 26G3/8 needle&#160;</td>
			<td style="width:205px;">e.g. Becton Dickinson &#38; Co.</td>
			<td style="width:119px;">309625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringes, 2 ml</td>
			<td style="width:205px;">e.g. B. Braun</td>
			<td style="width:119px;">7389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needles, 25G5/8 and 30G1/2&#160;</td>
			<td style="width:205px;">e.g. Becton Dickinson &#38; Co.,</td>
			<td style="width:119px;">305122, 305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Three way halve</td>
			<td style="width:205px;">e.g. B. Braun</td>
			<td style="width:119px;">16494 C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pertussis toxin, lyophilized in buffer</td>
			<td style="width:205px;">Enzo Life Sciences Inc.</td>
			<td style="width:119px;">BML-G100</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">M. tuberculosis H37 RA</td>
			<td style="width:205px;">BD Difco&#160;</td>
			<td style="width:119px;">231141</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
	</tbody>
</table>

myelin oligodendrocyte glycoprotein (mog35-55) induced experimental autoimmune encephalomyelitis (eae) in c57bl/6 mice

多发性硬化是一种中枢神经系统具有强大的神经变性成分的慢性神经炎症性脱髓鞘性疾病。而该疾病的确切病因还不清楚，自身反应性T淋巴细胞被认为在它的病理生理中发挥核心作用。 MS的治疗只是部分有效，至目前为止的研究工作继续扩大我们对疾病的病理生理学知识，并开发新的治疗策略。实验性自身免疫性脑脊髓炎（EAE）是最常见的动物模型中的MS共享许多临床和病理特征。有EAE模型反映人类的MS不同临床，免疫学和组织学方面的广泛多样性。在小鼠积极诱导EAE是具有强大的和可复制的结果最容易诱发模型。它特别适用于通过使用由自身免疫性neuroi质疑转基因小鼠研究中特定基因的药物或效果nflammation。因此，小鼠用CNS组织匀浆或髓鞘蛋白的肽。由于这些肽的低免疫原性的潜力，强佐剂的使用。 EAE的易感性和表型取决于所选择的抗原和啮齿动物品系。 C57BL / 6小鼠是最常用的应变为转基因小鼠和施工等等响应髓鞘少突胶质细胞糖蛋白（MOG）。免疫原性表位的MOG 35-55在完全弗氏佐剂（CFA）免疫前和百日咳毒素被暂停施加在免疫的当天和两天后。小鼠制定了“经典”自限性的单相EAE与内免疫接种后9-14天升弛缓性瘫痪。鼠标使用的是临床评分系统25-50天每天进行评估。特别考虑照顾回吐动物与EAE以及这种模式的应用潜力和局限性进行了讨论。

免疫接种后，小鼠都必须每日体重变化及临床症状进行评价。发病通常关联与减少的重量可能开始1-2天前EAE的症状是可见的。 EAE的临床症状通常是9日和14后之间的免疫接种开始。如病变主要定位至脊髓中MOG-EAE在C57BL / 6小鼠，它们通常在开发一个尾鳍喙图案主要肌肉运动症状。经免疫的小鼠的EAE症状的示例性图像（分值6）被描绘在图1A中 。一些非典型EAE症状也可能发生，如轧制轴向旋转的方式，弯腰驼背的外观或感觉过敏不属于经典的EAE中反映描绘评分表1，由于电机的症状是在这个模型的主要特点，这个分数确实给我们疾病严重程度的良好指标。不同的监测系统（ 如 0分-5或0-6）和更复杂的综合得分评估不同的赤字也已出版。请注意，小鼠有严重症状必须根据各自的机构动物护理和使用委员会，取出实验。小鼠表现出症状，在未来10-20天一般部分恢复。代表性的病程是描绘在图1B。有哪些感兴趣的评估结果参数EAE在不同的时间点。这常常可以发现一些典型试验是很简单的描述。在疾病最高，老鼠可以分离的免疫细胞与MOG 35-55肽在体外 （MOG召回检测）的再刺激后评估细胞因子的产生和扩散。免疫细胞也可从脑和小鼠脊髓与EAE症状分离和进一步处理， 例如通过流式细胞仪。可在疾病米进行的脊髓切片组织学分析特长为病灶负荷和脱髓鞘，而在后来的时间点标记为神经变性和神经元细胞死亡可能是感兴趣的。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51275/51275fig1highres.jpg" src="/files/ftp_upload/51275/51275fig1.jpg" width="600px" /> 图1代表的EAE的结果。的免疫C57BL / 6小鼠EAE与症状A.代表图片（EAE比分6）。在免疫后30天B.代表病程。数据显示为平均值和平均值的标准误差（N = 5）。 <a href="https://www.jove.com/files/ftp_upload/51275/51275fig1highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice at JoVE.com

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

实验性自身免疫性脑脊髓炎（EAE）是多发性硬化的一个建立的动物模型。 C57BL / 6小鼠用髓鞘少突胶质细胞糖蛋白（MOG）肽35-55（MOG 35-55），因此所造成的自身反应性免疫细胞，在中枢神经系统中的上行弛缓性麻痹。协议对疾病的诱导和监控将被讨论。

髓鞘少突胶质细胞糖蛋白（MOG<sub&gt; 35-55</sub&gt;）诱导实验性自身免疫性脑脊髓炎（EAE）的C57BL / 6小鼠

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

myelin-oligodendrocyte-glycoprotein-mog35-55-induced-experimental

Research

JoVE Journal

Immunology and Infection

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

81.1K 次观看.  University of Münster. 实验性自身免疫性脑脊髓炎（EAE）是多发性硬化的一个建立的动物模型。 C57BL / 6小鼠用髓鞘少突胶质细胞糖蛋白（MOG）肽35-55（MOG 35-55），因此所造成的自身反应性免疫细胞，在中枢神经系统中的上行弛缓性麻痹。协议对疾病的诱导和监控将被讨论。 

视频: Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

科研

免疫与感染

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

在实验性自身免疫性脑脊髓炎（EAE）抗性品系小鼠继转移和抗原挑战克服反应迟钝

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

简单而高效的生产和鼠标髓鞘少突胶质糖蛋白的纯化的实验性自身免疫性脑脊髓炎研究

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

副结核分枝杆菌在自身免疫性脑脊髓炎中的免疫原性增强

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

作者聚焦： 副结核分枝杆菌在增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原性中的辅助活性  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...